Iron is an abundant metal in the environment providing challenges to human health by contributing to envi- ronmental toxicity. Iron is a transition metal that exists in two pools within cells. Chelatable iron comprises free iron and iron loosely bound to anionic metabolites like ATP and citrate, whereas non-chelatable iron is tightly bound to ferritin, heme and iron-sulfur clusters. Chelatable iron promotes oxidative stress by catalyzing the Fen- ton reaction, which produces highly reactive hydroxyl radicals that damage DNA, proteins and membranes. Sub- stantial evidence implicates mitochondrial iron as an important contributor to toxicity, but the molecular pathways of mitochondrial iron uptake are controversial. The prevailing view is that mitoferrins 1 and 2 (Mfrn1/2), proteins localized in the mitochondrial inner membrane, are responsible for mitochondrial iron transport. However, previ- ous studies from ~40 years ago show that the classical electrogenic mitochondrial calcium uniporter also cata- lyzes uptake of Fe2+ but not Fe3+ driven by the mitochondrial membrane potential, a conclusion supported by our own recent studies in intact hepatocytes. Our preliminary pull-down and Duolink studies indicate a physical as- sociation between Mfrn2, the predominant isoform in non-erythropoietic cells, and MCU, the core protein of the uniporter complex, which brings us to the fundamental questions to be addressed by this proposal: 1) Does electrogenic mitochondrial Fe2+ and Ca2+ uptake occur via two independent pathways: one mediated by Mfrn1/2 and another by MCU; or does electrogenic uptake of both cations occur exclusively by one or the other carrier? 2) Alternatively, do Mfrn and the calcium uniporter act cooperatively in mediating up- take of both iron and calcium? Therefore, the studies proposed here are intended to address these questions utilizing Mfrn1/2 single knockout (KO) and double KO (DKO) hepatocytes and their wild type (WT) counterparts. In Aim 1, mitochondrial Fe2+ uptake will be measured in permeabilized mouse hepatocytes by confocal micros- copy using a newly developed mitochondria-specific Fe2+-indicating fluorophor, mitoferrofluor. In Aim 2, mito- chondrial Ca2+ uptake will be measured using Ca2+-indicating Fluo5N. If there are two independent pathways (#1 above), then Mfrn2 deficiency will prevent Fe2+ but not Ca2+ uptake, and MCU deficiency will prevent Ca2+ but not Fe2+ uptake, whereas if Mfrn2 and MCU reside in a single complex (#2), then the kinetics of both Fe2+ and Ca2 uptake should be altered by Mfrn2 deficiency, and both Fe2+ and Ca2 uptake will be blocked by MCU deficiency. The concept that MCU and Mfrn are essential for both Fe2+ and Ca2 electrogenic uptake is novel, innovative and paradigm-changing. The project will provide insights into an unexplored area of biology and fill an important gap in our understanding of the pathways involved in mitochondrial iron uptake and iron-dependent toxicities. Better understanding of this process will eventually lead to new more specific interventions against toxicities promoted by iron overload.